The present invention provides a method of inhibiting protein kinase C in a mammal having a tumor system which contains protein kinase C, which mammal is undergoing cytostatic therapy. The method comprises administering to the mammal a protein kinase C inhibitor.
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1. A method of inhibiting protein kinase C in a mammal having a tumor system which contains protein kinase C and which mammal is undergoing cytostatic therapy, said method comprising administering to said mammal an effective amount of a protein kinase C inhibiting compound selected from the group consisting of quercetin,ilmofosin, 4-hydroxy-7-methoxy-N,N,N-trimethyl-3,5,9-trioxa-4-phosphaheptacosan-1-ami
nium 4-oxide and staurosporin.
2. Method of
3. Method of
4. Method of
6. Method of
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This application is continuation of application Ser. No. 07/759,201, filed filed Sep. 11, 1991, now abandoned, which was a continuation of U.S. Ser. No. 07/395,698, filed Aug. 18, 1989, now abandoned.
The present invention is concerned with a method of inhibiting protein kinase C on a mammal having a tumor system which contains protein kinase C and which mammal is undergoing cytostatic therapy.
In particular, the present invention is concerned with combination preparations of protein kinase C inhibitors with cytostatically-active compounds.
In anti-neoplastic therapy, the use of chemotherapeutics has already long been a recognised and widely used treatment principle. These chemotherapeutics are used in order to destroy malign cells with uninhibited growth behaviour, whereas normal or healthy cells are to be damaged as little as possible. However, as chemotherapeutics there are used almost exclusively cytostatics which, in general, act non-specifically toxically on normal and malign cells and inhibit the growth of the cells. These cytostatics have a very narrow therapeutic breadth of use, which results in serious side effects.
Such side effects include, for example, haemorrhages, nausea, vomiting, dyspnoea, allergies, alopecias, heart muscle damage, heart rhythm disturbances, pericarditis, peripheral and central neuropathies, pain, nepthropathies, stomatitis, diarrhoea, fever, skin changes, infections, heart insufficiencies or changes of the state of consciousness.
Therefore, it is an object of the present invention to increase the action of chemotherapeutics without involving an increase of the toxic effects of these active materials. Medicaments are to be made available which bring about a reduction of the above-mentioned side effects in the case of the treatment with chemotherapeutics. It is thereby an object to increase the therapeutic breadth of use of these chemotherapeutics. In the case of cytostatics, the anti-tumour and anti-proliferative action is to be strengthened so that these cytostatics can be administered in smaller doses and thus a reduction or removal of the side effects brought about by these agents takes place.
In the meaning of the present invention, by the term "protein kinase C inhibitors" are to be understood those compounds which inhibit calcium- and phospholipid-dependent protein kinase C or the corresponding isoenzymes thereof in cell-free extracts or in intact cells (Nishizuka, Science, 233, 305-312/1986; Nature, 334, 662-665/1988). In this sense, the following compounds can, for example, be used: quercetin (3,3',4',5,7-pentahydrdoxyflavone; Horn, F., J. Biochem., 148, 533-538/1985), tamoxifen (O-Brien et al., Cancer Research, 45, 2462-2465/1985), staurosporin (Tamaoki, T. et al., Biochem. Biophys. Res. Comm., 135, 397-402/1986), ilmofosin, and ET-18-OCH3. With the help of the general process described in the following Example 1, it can readily be determined experimentally whether a compound acts as an inhibitor of protein kinase C and can be used in the sense of the present invention.
Ilmofosin and processes for the preparation thereof are known from European Patent Specification No. 0,050,327. The compound is there described as Example 33 with the systematic name 3-hexadecylmercapto-2-methoxypropanol-1 phosphoric acid monocholine ester. Ilmofosin belongs to the group of so-called alkyl-lysolecithin derivatives and is known as compound with cancerostatic properties.
ET-18-OCH3, the systematic name of which is 4-hydroxy-7-methoxy-N,N,N-trimethyl-3,5,9-trioxa-4-phosphaheptacosan-1-ami nium 4-oxide, is known from Federal Republic of Germany Patent Specification No. 26 19 686. This compound is there described as being an anti-tumour agent.
The cytostatic therapy involves the administration of chemotherapeutics. Amongst these are to be understood in the following substances foreign to the body which are suitable for and are used in order to damage or destroy micro-organisms, parasites (antibiotics) or rumour cells (cytostatics). Cytostatics and the derivatives thereof from the following groups of cytostatics are thereby to be especially mentioned: cyclophosphamide, chlorambucil, metal complex cytostatics, for example metal complexes of platinum, for example cis-diamminedichloroplatinum (II); methotrexate, 5-fluorouracil, doxorubicin, bleomycin, tamoxifen.
Anti-neoplastic active materials which are especially preferred according to the present invention include platinum complex compounds, for example, cis-dichlorodiammine-platinum (II) and (IV), mustargen and doxorubicin (adriamycin).
In special cases, it can also happen that a compound not only falls within the group of already known anti-neoplastic active materials in the meaning of the present invention but can also be assigned to the group of protein kinase C inhibitors. This is, for example, the case with ilmofosin and ET-18-OCH3. However, this does not exclude the possibility of combination with other anti-neoplastic active materials so long as at least one of the active material functions as a protein kinase C inhibitor.
The use of a combination therapy with help of the pharmaceutical preparations of the present invention offers the advantage of the synergistic strengthening of the action of the individual substances. The possibility of the reduction of the doses and thus of the toxicities of the individual substances in the case of simultaneous maintenance of the effectiveness of the combination of the individual substances is thereby provided for. Furthermore, a combination therapy of the above-mentioned individual therapy principles offers the possibility of overcoming cytostatic resistances, which includes not only substance group resistances but also multiple resistances (pleiotropic cytostatic resistance).
In the case of the use of the combination therapy, it is possible to administer the active materials in a so called fixed combination, i.e. in a single pharmaceutical formulation which contains both active materials or to choose a so called free combination in which the active materials, in the form of separate pharmaceutical formulations, can be administered simultaneously or also successively. Such combination preparations can be prepared according to known processes which are usual in galenical technology.
If the active materials are solids, then the active materials can be worked up by usual processes to give solid medicament preparations (tablets, pellets, compresses, gelatine capsules), for example by mixing both active materials with one another and, together with usual carrier and adjuvant materials, pressing to give, for example, tablets. However, it is also possible to make the active materials available, together with appropriate pharmaceutical adjuvants, separate from one another in packing units ready for sale, the packing unit thereby containing the two active materials in separate pharmaceutical formulations.
If the active materials are made available in the form of injection solutions, then these can contain the active material combinations in question in lyophilised form or already in final injectably dissolved form. However, in principle, it is also possible to make available a parenteral formulation for each active material in question in a packing unit so that the injection solutions can possibly be administered separately from one another. In the case of incompatibilities of the active materials with one another, this form of use is the preferred method.
In the case of the parenteral form of administration, the active materials can also be present in substance, possibly together with usual pharmaceutical adjuvant materials, for example in lyophilised form, which can be reconstituted or solubilised by the addition of conventional pharmaceutical injection media.
The pharmaceutical preparations are administered enterally or parenterally in liquid or solid form. All conventional forms of administration can thereby be used, for example tablets, capsules, dragees, syrups, solutions and suspensions. As injection medium, it is preferred to use water which contains the additives usual in the case of injection solutions, such as stabilising agents, solubilising agents and buffers. Additives of this kind include, for example, tartrate and citrate buffers, ethanol, complex formers, for example ethylenediamine-tetraacetic acid and the non-toxic salts thereof, as well as high molecular weight polymers, such as liquid polyethylene oxide, for viscosity regulation. Liquid carrier materials for injection solutions must be sterile and are preferably placed into ampoules. Solid carrier materials include, for example, mannitol, starch, lactose, silicic acids, higher molecular weight fatty acids, such as stearic acid, gelatine, agar-agar, calcium phosphate, magnesium stearate, animal and vegetable fats and solid high molecular weight polymers, such as polyethylene glycols. Compositions suitable for oral administration can, if desired, also contain flavouring and sweetening materials.
The dosaging can depend upon various factors, such as the mode of administration, species, age and individual state. The doses to be administered daily are about 0.05 no 100 mg./kg. body weight per individual component. In the case of a combination comprising cis-DDP and quercetin, a dosage of, for example, 1-10 mg/kg, especially 3 mg/kg, cis-DDP and 10-50 mg/kg, especially 20 mg/kg, quercetin can be used. The amount of the particular active material per form of administration can be from 5 to 1000 mg.
In the case of the combination preparations, the ratio between the active materials functioning as inhibitors of protein kinase C and the lipids, lipid analogues, cytostatics or inhibitors of phospholipases can vary within a very wide range. Thus, for example, molar ratios of from 1:1000 to 1000:1 are possible, depending upon the effectiveness of the active materials in question. In the case of a combination with cytostatics, a ratio of from 1:100 to 100:1 is preferred. In particular, in the case of the combination of ilmofosin or ET-18-OCH3 with cis-diamminedichloroplatinum (II), a ratio of from 1:50.to 50:1 can be used but preferably of from 1:1 to 50:1.
In the meaning of the present invention, the following combination preparations can be mentioned by way of example:
| ______________________________________ |
| protein kinase C |
| anti-neoplastic |
| molar ratio |
| inhibitor A compound B A:B |
| ______________________________________ |
| quercetin cis-DDP about 100:1 |
| quercetin mustargen about 800:1 |
| tamoxifen cis-DDP about 10:1 |
| staurosporin cis-DDP about 1:100 |
| ilmofosin cis-DDP about 10:1 |
| ET-18-OCH3 |
| cis-DDP about 10:1 |
| ______________________________________ |
The following Examples demonstrate the synergistic action of some representative combination preparations and exemplify the principles of the present invention.
General process for investigating compounds with regard to their function as inhibitor of protein kinase C'.
a) Reagents
Horse serum and DMEM (Dulbecco's modified minimal essential medium) were obtained from Boehringer Mannheim GmbH, FRG. Cis-diamminedichloroplatinum (II) was obtained from Homburg Pharma, Frankfurt. [gamma-32 P]-ATP (10 Ci/mmol) and 32 P-orthophosphate were obtained from Radiochemical Centre, Amersham, U.K.; GF/F filters and DE-52-cellulose were obtained from Whatman, Clifton N.J. (U.S.A.), leupeptin, aprotinin, quercetin (3,3',4',5,7-pentahydroxyflavone), tamoxifen, 12-O-tetradecanoylphorbol 13-acetate (TPA), β-glycerophosphate, histone HI (Type III S), 3-N-morpholinopropane sulphonylic acid (MOPS), L-alpha-phosphatidyl-L-serine and 1,2-sn-diolein were obtained from Sigma, M unchen, FRG, and protein kinase C was obtained from Merck, Darmstadt, FRG. Tris-(hydroxymethyl)-aminomethane (TRIS-HCl), ethyleneglycol-bis-(aminoethyl)-tetraacetic acid (EGTA), sodium dodecyl sulphate (SDS) and Triton X-100 were obtained from Serva, Heidelberg, mustargen (HN2) was obtained from Aldrich, Steinheim and staurosporin originated from Prof. Matter, Ciba-Geigy, Basel, Switzerland.
Ilmofosin was obtained from Boehringer Mannheim GmbH and was synthesised as described by Bosies et al. (Lipids, 22, 947-951/1987). For the investigations, there was used a parent solution of 100 μg./ml. ilmofosin in DMEM+10% foetal calf serum (FCS). The parent solution was stored at 4°C
Doxorubicin (adriamycin, Adriablastin®) was obtained from Farmitalia/Carlo Erba GmbH, Freiburg, FRG.
b) Inhibition of protein kinase C (PK-C): in vitro method.
Protein kinase C was enriched from Walker cells by chromatography on DEAE-cellulose of the cell extracts by the method described by Kreutter et al. (J. Biol. Chem., 260, 5979-5984/1985). However, to the extracts was additionally added 1 mM phenylmethanesulphonyl chloride, 20 μg./ml. leupeptin and 2 μg./ml. aprotinin. The protein kinase C activity was determined by measurement of the 32 P incorporation of [gamma-32 P]-ATP into H1 histone according to the method of Fabbro et al. (Arch. Biochem. Biophys., 239, 102-111/1985). The reaction mixture (125 μl.) contained 0.5 μCi [gamma-32 P]-ATP, 40 mM Tris-HCl (pH 7.4), 1 mM calcium chloride, 700 μM EGTA, 50 μg. histone, 6.75 μg. L-alpha-phosphatidyl-L-serine and 0.675 μg. 1,2-s,n-diolein. The reaction time was 10 minutes at 32°C The enzyme reaction was stopped by the addition of 1 ml. 20% trichloroacetic acid (w/v). The protein was precipitated out on Whatman GF/F filter paper and counted with the help of a liquid scintillation counter.
c) Inhibition of protein kinase C' (PK-C')--in vivo method--Phosphorylation of ribosomal protein S6.
Cells were cultured in DMEM with 0.5% horse serum (v/v) over a period of time of 15 hours and then incubated in phosphate-free medium. After 1 hour, 4 μCi/ml. 32 P-orthophosphate and, after a further 30 minutes, 0.5 μM TPA (12-O-tetradecanoylphorbol 13-acetate) and 50 μM quercetin were added thereto. The cells were lysed in 50 mM Tris-HCl (pH 7.5), 2.5 mM potassium chloride, 5 mM magnesium chloride, 0.33M saccharose, 1% Triton X-100 (v/v), 1 mM phenylmethanesulphonyl chloride, 20 μg./ml. leupeptin, 2 μg./ml. aprotinin and 80 mM β-glycerophosphate. After 10 minutes at 0°C, the lysate was centrifuged at 30,000 g for 10 minutes. The pellets were washed once by resuspension with the lysis buffer and subsequently centrifuged at 30,000 g. The supernatants were combined and centrifuged at 100,000 g. for 3 hours. The pellets were resuspended in 8M urea solution and boiled for 10 minutes. The extracts were analysed by one-dimensional SDS gel electrophoresis in 15% polyacrylamide gels (Laemmli, U.K., Nature, 227, 680-685/1970). After blotting the gel on to nitrocellulose, the S6 protein was identified by the addition of anti-S6-antiserum.
PAC Quercetin as Inhibitor of Protein Kinase CAs was described in more detail in Example 1, the influence of quercetin on protein kinase C and on the multiplication of Walker carcinoma cells in culture was investigated. Quercetin was solubilised in dimethyl sulphoxide (DMSO). For the batch mixing, the solution in DMSO was made up to a DMSO end concentration of 1%. An equivalent amount of pure DMSO was added to the control groups. The influence of quercetin on the cell multiplication was investigated by the addition of the active material in DMSO to the culture medium up to an end concentration of 0.1%. The cells were cultured in the presence of the active material for 48 hours. The control group only received pure DMSO. 100% of the protein kinase C activity corresponds to 44.8 pmol/min. 32 P transferred to H1.
From the data obtained, it follows that quercetin functions as an inhibitor of protein kinase C (IC50 =25 μM).
PAC Tamoxifen as Inhibitor of Protein Kinase CAnalogously to the description in Example 2, the influence of tamoxifen on protein kinase C was investigated. From the data obtained, the IC50 value could be determined as being 11.20 μM.
PAC Staurosporin as Inhibitor of Protein KinaseAnalogously to the description in Example 2, the influence of staurosporin on protein kinase C was investigated. The IC50 value was determined as being 0.048 μM.
PAC Ilmofosin as Inhibitor of Protein Kinase CAnalogously to the description in Example 2, the effect of ilmofosin on protein kinase C was investigated. The IC50 value was 20 μM.
PAC ET-18-OCH3 as Inhibitor of Protein Kinase CAnalogously to the description in Example 2, the influence of ET-18-OCH3 on protein kinase C was investigated. In the same way as ilmofosin, ET-18-OCH3 inhibits protein kinase C (IC50 =24.8 μM).
PAC General Process for the Determination of the Synergistic Effect of a Combination of Protein Kinase C Inhibitor and an Anti-neoplastic Active Materiala) Walker carcinoma cells from rats were cultured in suspensions of DMEM (Dulbecco's modified minimal essential medium) with a 10% portion (v/v) of horse serum and 25 mM MOPS buffer (pH 7.35 at 20°C) at a temperature of 36.8°C Dose-action curves for individual active materials or active material combinations were obtained by the addition of corresponding active materials to a suspension of Walker cells (105 cells/ml.). After an incubation time of 48 hours, the cells were counted with the help of an electronic counter (Coulter Electronics, Luton, U.K.). The multiplication of the cells (M) was calculated according to the following formula:
M=(Tt -T0)/(Ct -C0)*100,
in which C stands for the untreated control cells, T signifies the number of treated cells and the indices 0 and t indicate the number of the cells at the time point 0 and after 48 h.
The synergistic effect of the active material combination was determined by the method described by Chou and Talalay (Eur. J. Biochem., 115, 207-216/1981 and Advances Enzyme Regul., 22, 27-54/1984). The data used for the calculation originated from at least three different experiments. The calculation programme was obtained from Elsevier Biosoftware, Cambridge, U.K. (dose effect analysis with microcomputers).
b) [3H]-Thymidine incorporation.
The cytostatic or cytotoxic effect of the active materials or active material combinations on Meth Afibrosarcoma cells was investigated in vitro on the basis of the reduced incorporation of [3H]-thymidine. The cells were suspended in DMEM, 10% FCS, 50 μM 2-mercaptoethanol, 100 U/ml. penicillin and 100 μg./ml. streptomycin up to an end concentration of 5×104 /ml. in the absence of the active materials. The active materials were added to the cells in an end volume of 20 μl. Per concentration, 6 cultures of 0.2 ml. were used and incubated in microtitre plates in a moist atmosphere. The cultures were pulsed for 3 hours with 1 μCi (27 kBq/cell) [methyl-3 H]-thymidine (specific activity 5 Ci/mmol). The samples were subsequently collected and washed several times. The filter plates used were dried and transferred to scintillation test tubes. The radio-active incorporation was measured by the addition of Rotiscint.
PAC Synergistic Effect of Quercetin and Cis-diamminedichloroplatinum (II) (cis-DDP)As described in Example 7a, the influence of cis-DDP, quercetin and a mixture of quercetin/cis-DDP (molar ratio 100:1) on Walker sarcoma cells from rats was investigated. The cells were cultured in the presence of the active materials in question for a period of 48 hours. The result is given in the following Table 1.
PAC Synergistic Combination of Quercetin and MustargenAs described in Example 7a, the influence of quercetin, mustargen and a mixture of quercetin/mustargen (molar ratio 800:1) on Walker sarcoma cells from rats was investigated. The result is given in the following Table 1.
PAC Synergistic Combination of Tamoxifen and Cis-DDPAs described in Example 7a, the influence of tamoxifen, cis-DDP and a mixture of tamoxifen/cis-DDP (molar ratio 10:1) on Walker sarcoma cells from rats was investigated. The result is given in the following Table 1.
PAC Synergistic Combination of Staurosporin and Cis-DDPAs described in Example 7a, the influence of staurosporin, cis-DDP and a mixture of staurosporin/cis-DDP (molar ratio 1:100) on Walker sarcoma cells from rats was investigated. The result is given in the following Table 1.
PAC Synergistic Combination of Ilmofosin and Cis-DDPAs described in Example 7a, the influence of ilmofosin, cis-DDP and a mixture of ilmofosin/cis-DDP (molar ratio 10:1) on Walker sarcoma cells from rats was investigated. The result is given in the following Table 1.
PAC Synergistic Combination of ET-18-OCH3 and Cis-DDPAs described in Example 7a, the influence of ET-18-OCH3, cis-DDP and a mixture of ET-18-OCH3 /cis-DDP (molar ratio 10:1) on Walker sarcoma cells from rats was investigated. Similar to what was described in Example 12, in this case, too, a synergistic action was ascertained (see Table 1).
| TABLE 1 |
| __________________________________________________________________________ |
| Inhibition of the cellular replication and strengthening |
| of the anti-proliferative effect of cis-DDP by inhibition |
| of protein kinase C; summary of the IC50 values |
| IC50 [μM](1) |
| inhibition of the(1) |
| cell profiler- |
| protein |
| cell pro- |
| ation in com- |
| nature(2) |
| inhibitor kinase C |
| liferation |
| bination with |
| of the |
| (example) IC50 [μM] |
| IC50 [μM] |
| cis-DDP activity |
| __________________________________________________________________________ |
| quercetin (2,89) |
| 25 23 3.8 synergism |
| tamoxifen (3,10) |
| 11.20 12.44 2.24 synergism |
| staurosporin4,11) |
| 0.048 0.4 0.004 synergism |
| ilmfosin (5,12) |
| 0,56 20 2 synergism |
| ET-18-OCH3 (6,13) |
| 24.8 5.8 1.7 synergism |
| cis-DDP >1000 0.23 -- -- |
| __________________________________________________________________________ |
| (1) By the IC50 value is to be understood that concentration of |
| the inhibitor at which a 50% inhibition of the protein kinase C or of the |
| cell proliferation is reached. |
| (2) The calculation basis with regard to the synergistic effect took |
| place according to the method of Chou and Talalay (Advances Enzyme Regul. |
| 22, 27-54/1984). |
The IC50 values for a combination of ilmofosin with CDDP or doxorubicin were determined in the manner described above in Example 7b). The results obtained are shown in the following Table 2.
| TABLE 2 |
| ______________________________________ |
| Inhibition of tumour cell proliferation |
| inhibitor |
| anti-neoplastic |
| PK-C active material ratio IC50 |
| A B A:B [μg./ml.] |
| ______________________________________ |
| ilmofosin |
| CDDP 100:1 1.09 |
| ilmofosin |
| doxorubicin 100:1 1.11 |
| ilmofosin |
| -- -- 1.06 |
| ______________________________________ |
The compounds A and B selected as active materials can be used in various galenical formulations. The following Examples concern galenical compositions which contain an active material designated as A as protein kinase C inhibitor and an anti-neoplastic active material designated as B.
| ______________________________________ |
| a) Tablets |
| mixture I mixture II |
| ______________________________________ |
| active material A |
| 50 mg. active material B |
| 50 mg. |
| starch 180 mg. silicon dioxide |
| 100 mg. |
| magnesium stearate |
| 20 mg. lactose 100 mg. |
| Aerosil 5 mg. |
| ______________________________________ |
Mixtures I and II are dry or moist granulated separate from one another. Subsequently, they are mixed with one another with the addition of 5 mg. talc and pressed into tablets.
| ______________________________________ |
| b) Capsules |
| mixture I mixture II |
| ______________________________________ |
| active material A |
| 50 mg. active material B |
| 200 mg. |
| lactose 110 mg. polyvinyl- 10 mg. |
| pyrrolidone |
| maize starch |
| 20 mg. maize starch |
| 100 mg. |
| gelatine 8 mg. Ceetina HR 10 mg. |
| magnesium stearate |
| 12 mg. |
| ______________________________________ |
Separately from one another, the two mixtures A and B are granulated in the usual manner. The two granulates are mixed with one another in a mixer in the given mixing ratio and the powder mixed in the mixer with talc. Subsequently, the mixture obtained is filled into hard gelatine capsules in a conventional machine.
c) Injection solutions (i.m. or i.v.)
An injection solution ready for intravenous (i.v.) injection contains:
| ______________________________________ |
| active material A |
| 50 mg. |
| active material B |
| 100 mg. |
| sodium chloride 20 mg. |
| sodium acetate 6 mg. |
| distilled water ad |
| 5 ml. |
| ______________________________________ |
An injection solution ready for intramuscular (i.m.) injection contains:
| ______________________________________ |
| active material A |
| 100 mg. |
| active material B |
| 100 mg. |
| benzyl benzoate 1 g. |
| injection oil 5 ml. |
| ______________________________________ |
Bosies, Elmar, Hofmann, Johann, Herrmann, Dieter, Grunicke, Hans H.
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